The Notch locus of D. melanogaster is required early in embryogenesis for proper differentiation of the ectoderm. A complex complementation pattern is also found for mutations defining the gene. In an attempt to understnd the molecular basis of Notch function, DNA from the locus has been cloned and characterized in considerable detail. The Notch locus corresponds to a 37-kb transcription unit that generates polyA+ RNA about 11.7 kb in length. Most mutations at the Notch locus appear to alter mRNA coding sequences and therefore should produce aberrant Notch protein. In this proposal we will extend this analysis as follows: 1) Notch locus DNA has been used to generate E. coli hybrid plasmids that synthesize Notch peptides. These will be used to generate antibody to the in vivo Notch protein so that its structure and tissue distribution in mutant and wild-type flies can be assessed. 2) The structure and tissue distribution of Notch RNA in mutant and wild-type flies will be determined in order to decide how several DNA insertions near intron-exon boundaries generate mutant phenotypes. 3) Certain copia-like transposable elements produce tissue-specific, mutant phenotypes by interrupting intervening sequences. The molecular basis for their modulation of Notch locus expression will be explored by determining if the expression of each element corresponds temporally and spatially with suppression of Notch locus activity. We will also attempt to generate revertants of these insertion mutants. 4) The chromosomal intervals adjoining the Notch locus are heavily transcribed, but the functions of these DNA sequences are not apparent from previous genetic analyses. The structures of these transcription units and their precise boundaries with respect to the Notch locus will be mapped. 5) An attempt will be made to transform mutant flies with cloned Notch DNA and its derivatives. 6) A physical analysis of the Notch locus in a distantly related species of Drosophila will be undertaken.